In silico and in vitro assessment of drugs potentially causing adverse effects by inhibiting CYP17A1.

Cytochrome P450 enzymes (CYPs) play a crucial role in the metabolism and synthesis of various compound classes. While drug-metabolizing CYP enzymes are frequently investigated as anti-targets, the inhibition of CYP enzymes involved in adrenal steroidogenesis is not well studied. The steroidogenic enzyme CYP17A1 is a dual-function enzyme catalyzing hydroxylase and lyase reactions relevant for the biosynthesis of adrenal glucocorticoids and androgens. Inhibition of CYP17A1-hydroxylase leads to pseudohyperaldosteronism with subsequent excessive mineralocorticoid receptor activation, hypertension and hypokalemia. In contrast, specific inhibition of the lyase function might be beneficial for the treatment of prostate cancer by decreasing adrenal androgen levels. This study combined in silico and in vitro methods to identify drugs inhibiting CYP17A1. The most potent CYP17A1 inhibitors identified are serdemetan, mocetinostat, nolatrexed, liarozole, and talarozole. While some of these drugs are currently under investigation for the treatment of various cancers, their potential for the treatment of prostate cancer is yet to be explored. The DrugBank database was screened for CYP17A1 inhibitors, to increase the awareness for the risk of drug-induced pseudohyperaldosteronism and to highlight drugs so far unknown for their potential to cause side effects resulting from CYP17A1 inhibition.

A battery of in silico models application for pesticides exerting reproductive health effects: Assessment of performance and prioritization of mechanistic studies.

Given the high attention to endocrine disrupting chemicals (EDC), there is an urgent need for the development of rapid and reliable approaches for the screening of large numbers of chemicals with respect to their endocrine disruption potential. This study aimed at the assessment of the correlation between the predicted results of a battery of in silico tools and the reported observed adverse effects from in vivo reproductive toxicity studies. We used VirtualToxLab (VTL) software and the EndocrineDisruptome (ED) online tool to evaluate the binding affinities to nuclear receptors of 17 pesticides, 7 of which were classified as reprotoxic substances under Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP). Then, we aligned the results of the in silico modelling with data from ToxCast assays and in vivo reproductive toxicity studies. We combined results from different in silico tools in two different ways to improve the characteristics of their predictive performance. Reproductive toxicity can be caused by various mechanisms; however, in this study, we demonstrated that the use of a battery of in silico tools for assessing the binding to nuclear receptors can be useful for identifying hazardous compounds and for prioritizing further studies.

Assessment of the inhibitory potential of anabolic steroids towards human AKR1D1 by computational methods and in vitro evaluation.

Exposure to xenobiotics can adversely affect biochemical reactions, including hepatic bile acid synthesis. Bile acids are essential for dissolving lipophilic compounds in the hydrophilic environment of the gastrointestinal tract. The critical micellar concentration of bile acids depends on the Δ4-reduction stereochemistry, with the 3-oxo-5ß-steroid-Δ4-dehydrogenase (AKR1D1) introducing the cis ring A/B conformation. Loss-of-function mutations in AKR1D1 cause hepatic cholestasis, which, if left untreated can progress into steatosis and liver cirrhosis. Furthermore, AKR1D1 is involved in clearing steroids with an A-ring Δ4-double bond. Here, we tested whether anabolic-androgenic steroids (AAS), often taken off-label at high doses, might inhibit AKR1D1, thereby potentially causing hepatotoxicity. A computational molecular model was established and used for virtual screening of the DrugBank database consisting of 2740 molecules, yielding mainly steroidal hits. Fourteen AAS were selected for in vitro evaluation, as such compounds can reach high hepatic concentrations in an abuse situation. Nandrolone, clostebol, methasterone, drostanolone, and methenolone inhibited to various extent the AKR1D1-mediated reduction of testosterone. Molecular modeling suggests that 9 out of 14 investigated AAS are competitive inhibitors. Moreover quantum mechanical calculations show that nadrolone and clostebol are substrates of AKR1D1 with different activation energy barriers for the hydrogen transfer from cofactor to the C5 position affecting their turnover. In this multidisciplinary approach, we established a molecular model of AKR1D1, identified several AAS as inhibitors, and described their binding mode. This approach may be applied to study other classes of inhibitors including non-steroidal compounds.

Evaluating the food safety and risk assessment evidence-base of polyethylene terephthalate oligomers: A systematic evidence map.

BACKGROUND: The presence of polyethylene terephthalate (PET) oligomers in food contact materials (FCMs) is well-documented. Consumers are exposed through their migration into foods and beverages; however, there is no specific guidance for their safety evaluation. OBJECTIVES: This systematic evidence map (SEM) aims to identify and organize existing knowledge and associated gaps in hazard and exposure information on 34 PET oligomers to support regulatory decision-making. METHODS: The methodology for this SEM was recently registered. A systematic search in bibliographic and gray literature sources was conducted and studies evaluated for inclusion according to the Populations, Exposures, Comparators, Outcomes, and Study type (PECOS) framework. Inclusion criteria were designed to record hazard and exposure information for all 34 PET oligomers and coded into the following evidence streams: human, animal, organism (non-animal), ex vivo, in vitro, in silico, migration, hydrolysis, and absorption, distribution, metabolism, excretion/toxicokinetics/pharmacokinetics (ADME/TK/PK) studies. Relevant information was extracted from eligible studies and synthesized according to the protocol. RESULTS: Literature searches yielded 7445 unique records, of which 96 were included. Data comprised migration (560 entries), ADME/TK/PK-related (253 entries), health/bioactivity (98 entries) and very few hydrolysis studies (7 entries). Cyclic oligomers were studied more frequently than linear PET oligomers. In vitro results indicated that hydrolysis of cyclic oligomers generated a mixture of linear oligomers, but not monomers, potentially allowing their absorption in the gastrointestinal tract. Cyclic dimers, linear trimers and the respective smaller oligomers exhibit physico-chemical properties making oral absorption more likely. Information on health/bioactivity effects of oligomers was almost non-existent, except for limited data on mutagenicity. CONCLUSIONS: This SEM revealed substantial deficiencies in the available evidence on ADME/TK/PK, hydrolysis, and health/bioactivity effects of PET oligomers, currently preventing appropriate risk assessment. It is essential to develop more systematic and tiered approaches to address the identified research needs and assess the risks of PET oligomers.

Evaluating the food safety and risk assessment evidence-base of polyethylene terephthalate oligomers: Protocol for a systematic evidence map.

BACKGROUND: Polyethylene terephthalate (PET) oligomers are ubiquitous in PET used in food contact applications. Consumer exposure by migration of PET oligomers into food and beverages is documented. However, no specific risk assessment framework or guidance for the safety evaluating of PET oligomers exist to date. AIM: The aim of this systematic evidence map (SEM) is to identify and organize existing knowledge clusters and associated gaps in hazard and exposure information of PET oligomers. Research needs will be identified as an input for chemical risk assessment, and to support future toxicity testing strategies of PET oligomers and regulatory decision-making. SEARCH STRATEGY AND ELIGIBILITY CRITERIA: Multiple bibliographic databases (incl. Embase, Medline, Scopus, and Web of Science Core Collection), chemistry databases (SciFinder-n, Reaxys), and gray literature sources will be searched, and the search results will be supplemented by backward and forward citation tracking on eligible records. The search will be based on a single-concept PET oligomer-focused strategy to ensure sensitive and unbiased coverage of all evidence related to hazard and exposure in a data-poor environment. A scoping exercise conducted during planning identified 34 relevant PET oligomers. Eligible work of any study type must include primary research data on at least one relevant PET oligomer with regard to exposure, health, or toxicological outcomes. STUDY SELECTION: For indexed scientific literature, title and abstract screening will be performed by one reviewer. Selected studies will be screened in full-text by two independent reviewers. Gray literature will be screened by two independent reviewers for inclusion and exclusion. STUDY QUALITY ASSESSMENT: Risk of bias analysis will not be conducted as part of this SEM. DATA EXTRACTION AND CODING: Will be performed by one reviewer and peer-checked by a second reviewer for indexed scientific literature or by two independent reviewers for gray literature. SYNTHESIS AND VISUALIZATION: The extracted and coded information will be synthesized in different formats, including narrative synthesis, tables, and heat maps. SYSTEMATIC MAP PROTOCOL REGISTRY AND REGISTRATION NUMBER: Zenodo: https://doi.org/10.5281/zenodo.6224302.

Migration Modeling as a Valuable Tool for Exposure Assessment and Risk Characterization of Polyethylene Terephthalate Oligomers.

Polyethylene terephthalate (PET) is one of the most widely used food contact materials due to its excellent mechanical properties and recyclability. Migration of substances from PET and assessment of compliance are usually determined by experimental testing, which can be challenging depending on the migrants of interest. Low concentrations and missing reference standards, among other factors, have led to inadequate investigation of the migration potential of PET oligomers. Migration modeling can overcome such limitations and is therefore a suitable starting point for exposure and risk assessment. In this study, the activation energy-based (EA) model and the AP model were used to systematically evaluate the migration potential of 52 PET oligomers for 12 different application scenarios. Modeling parameters and conditions were evaluated to investigate their impact and relevance on the assessment of realistic exposures. Obtained results were compared with safety thresholds known from the concept of toxicological thresholds of concern. This allowed the evaluation and identification of oligomers and/or applications where migration or exposure levels may be associated with a potential risk because they exceed these safety thresholds. Overall, this study demonstrated that migration modeling can be a high-throughput, fast, flexible, and suitable approach for comprehensive exposure assessment.

Evaluation of two in vitro assays for tumorigenicity assessment of CRISPR-Cas9 genome-edited cells.

Off-target editing is one of the main safety concerns for the use of CRISPR-Cas9 genome editing in gene therapy. These unwanted modifications could lead to malignant transformation, which renders tumorigenicity assessment of gene therapy products indispensable. In this study, we established two in vitro transformation assays, the soft agar colony-forming assay (SACF) and the growth in low attachment assay (GILA) as alternative methods for tumorigenicity evaluation of genome-edited cells. Using a CRISPR-Cas9-based approach to transform immortalized MCF10A cells, we identified PTPN12, a known tumor suppressor, as a valid positive control in GILA and SACF. Next, we measured the limit of detection for both assays and proved that SACF is more sensitive than GILA (0.8% versus 3.1% transformed cells). We further validated SACF and GILA by identifying a set of positive and negative controls and by testing the suitability of another cell line (THLE-2). Moreover, in contrast to SACF and GILA, an in vivo tumorigenicity study failed to detect the known tumorigenic potential of PTPN12 deletion, demonstrating the relevance of GILA and SACF in tumorigenicity testing. In conclusion, SACF and GILA are both attractive and valuable additions to preclinical safety assessment of gene therapy products.

Molecular mechanisms of posaconazole- and itraconazole-induced pseudohyperaldosteronism and assessment of other systemically used azole antifungals.

Recent reports described cases of severe hypertension and hypokalemia accompanied by low renin and aldosterone levels during antifungal therapy with posaconazole and itraconazole. These conditions represent characteristics of secondary endocrine hypertension caused by mineralocorticoid excess. Different mechanisms can cause mineralocorticoid excess, including inhibition of the adrenal steroidogenic enzymes CYP17A1 and CYP11B1, inhibition of the peripheral cortisol oxidizing enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) or direct activation of the mineralocorticoid receptor (MR). Compared to previous experiments revealing a threefold more potent inhibition of 11ß-HSD2 by itraconazole than with posaconazole, the current study found sevenfold stronger CYP11B1 inhibition by posaconazole over itraconazole. Both compounds most potently inhibited CYP11B2. The major pharmacologically active itraconazole metabolite hydroxyitraconazole (OHI) resembled the effects of itraconazole but was considerably less active. Molecular modeling calculations assessed the binding of posaconazole, itraconazole and OHI to 11ß-HSD2 and the relevant CYP enzymes, and predicted important interactions not formed by the other systemically used azole antifungals, thus providing an initial explanation for the observed inhibitory activities. Together with available clinical observations, the presented data suggest that itraconazole primarily causes pseudohyperaldosteronism through cortisol-induced MR activation due to 11ß-HSD2 inhibition, and posaconazole by CYP11B1 inhibition and accumulation of the mineralocorticoids 11-deoxycorticosterone and 11-deoxycortisol because of hypothalamus-pituitary-adrenal axis (HPA) feedback activation. Therapeutic drug monitoring and introduction of upper plasma target levels may help preventing the occurrence of drug-induced hypertension and hypokalemia. Furthermore, the systemically used azole antifungals voriconazole, isavuconazole and fluconazole did not affect any of the mineralocorticoid excess targets, offering alternative therapeutic options.

Identification of the fungicide epoxiconazole by virtual screening and biological assessment as inhibitor of human 11ß-hydroxylase and aldosterone synthase.

Humans are constantly exposed to a multitude of environmental chemicals that may disturb endocrine functions. It is crucial to identify such chemicals and uncover their mode-of-action to avoid adverse health effects. 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) catalyze the formation of cortisol and aldosterone, respectively, in the adrenal cortex. Disruption of their synthesis by exogenous chemicals can contribute to cardio-metabolic diseases, chronic kidney disease, osteoporosis, and immune-related disorders. This study applied in silico screening and in vitro evaluation for the discovery of xenobiotics inhibiting CYP11B1 and CYP11B2. Several databases comprising environmentally relevant pollutants, chemicals in body care products, food additives and drugs were virtually screened using CYP11B1 and CYP11B2 pharmacophore models. A first round of biological testing used hamster cells overexpressing human CYP11B1 or CYP11B2 to analyze 25 selected virtual hits. Three compounds inhibited CYP11B1 and CYP11B2 with IC50 values below 3 µM. The most potent inhibitor was epoxiconazole (IC50 value of 623 nM for CYP11B1 and 113 nM for CYP11B2, respectively); flurprimidol and ancymidol were moderate inhibitors. In a second round, these three compounds were tested in human adrenal H295R cells endogenously expressing CYP11B1 and CYP11B2, confirming the potent inhibition by epoxiconazole and the more moderate effects by flurprimidol and ancymidol. Thus, the in silico screening, prioritization of chemicals for initial biological tests and use of H295R cells to provide initial mechanistic information is a promising strategy to identify potential endocrine disruptors inhibiting corticosteroid synthesis. A critical assessment of human exposure levels and in vivo evaluation of potential corticosteroid disrupting effects by epoxiconazole is required.

The Binding of Human IgG to Minipig FcγRs - Implications for Preclinical Assessment of Therapeutic Antibodies.

PURPOSE: The Göttingen minipig is a relevant non-rodent species for regulatory toxicological studies. Yet, its use with therapeutic antibodies has been limited by the unknown binding properties of human immunoglobulins (huIgG) to porcine Fc gamma receptors (poFcγR) influencing safety and efficacy readouts. Therefore, knowing IgG-FcγR interactions in the animal model is a prerequisite for the use of minipigs in preclinical safety and efficacy studies with therapeutic antibodies. METHODS: Here, we describe the cloning and expression of poFcγRs and their interactions with free and complexed human therapeutic IgG1 by surface plasmon resonance and flow cytometry. RESULTS: We show here that poFcγRIa, poFcγRIIa, and poFcγRIIb bind huIgG1 antibodies with comparable affinities as corresponding huFcγRs. Importantly, poFcγRs bind huIgG immune complexes with high avidity, thus probably allowing human-like effector functions. However, poFcγRIIIa binds poIgG1a but not to huIgG1. CONCLUSIONS: The lack of binding of poFcγRIIIa to huIgG1 might cause underestimation of FcγRIIIa-mediated efficacy or toxicity as mediated by porcine natural killer cells. Therefore, the suitability of minipigs in preclinical studies with human therapeutic antibodies has to be assessed case by case. Our results facilitate the use of Göttingen minipigs for assessment of human therapeutic antibodies in preclinical studies.